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Degradation of myelin basic protein (MBP) in human man myelin was monitored by electroimmunoblotting. Problems of variation between, as well as within, electroimmunoblots were overcome by the introduction of an internal standard in each sample, thus allowing reproducible quantification of MBP. The Ca2+-dependent protease acting on MBP was active at endogenous levels of Ca2+ (congruent to 300 micrograms/g myelin) and was inhibited in the presence of Ca2+ chelators. Extensive degradation of MBP occurred rapidly in the presence of added Ca2+, reaching a plateau after a 1 h incubation (80-85% degradation). The proteolytic activity was not enhanced in the presence of 2-mercaptoethanol. It was most active at neutral pH and at temperatures approaching physiological conditions. No difference was observed between proteolytic activities of control and multiple sclerotic myelin. It is suggested that fluctuations in the accessibility of free Ca2+ to the protease may lead to the regulation of Ca2+-activated myelinolysis.

To carry out the immunological experiments on the serum of Multiple Sclerosis (MS) patients, based on a correlation between Borrelia burgdorferi infection and contracting MS autoimmune disease the outer surface protein D (OspD) of the bacterium was expressed and purified. A clone containing the OspD gene in pET11a expression vector under the control of T7 promoter was transformed to the bacterial host BL21 (DE3). Some of the colonies were selected for IPTG-induced expression. The colony with the highest amount of OspD was selected for large-scale expression. Large-scale protein purification was performed by the reversed phase HPLC with a C4 preparative column; ultimately, the expressed purified protein was confirmed by the Western blot technique.

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coli as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, Por = 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Serine proteases are thought to he involved in the initial attack on sheep skin by Dermatophilus congolensis and are obvious antigens for inclusion in a vaccine to prevent lumpy wool disease (dermato-philosis). Degenerate primers were designed after alignment of seven bacterial serine proteases. Inosine was incorporated into the primers at positions of three- and four-base redundancy, and this reduced the complexity of the primer mixtures from several thousand to sixteen different sequences for each primer. The primers were validated by production and sequencing of amplicons from serine protease genes in Bacillus suhtilis and Serratia marcescens. The primers were used with heat-soaked polymerase chain reaction (PCR) to produce amplicons from two D. congolensis strains, AG and MB. In the amplicon codons for arginine, rather than the expected serine, were found where inosine was used for both the first and third positions for a codon in the primer. A search with the deduced amino acid sequences of the amplicons showed significant similarity to a keratinase and other serine proteases from various organisms. Similarity was most apparent around the active site residues and other essential secondary structural elements.

The polymerase chain reaction method (PCR) was used to detect feline immunodeficiency virus proviral DNA in peripheral blood mononuclear cells (PBMC) of a group of 8 experimentally infected cats. The proportion of PBMC containing provirus was determined from 6 to 32 weeks post inoculation (p.i.) by performing PCR on serially diluted samples of PBMC. Primers from the p15 and p24 regions of the gag gene were used and Southern hybridization using an end-labelled probe was required to confirm primer-specific products. Provirus was detected in 5 of 8 cats by 6 weeks p.i. in 50000 PBMC, and in all 8 infected cats by 8 weeks p.i. Provirus was not detected in PBMC from any of 3 FIV negative cats. The proportion of PBMC containing provirus in individual cats ranged from 1 in 70 to 1 in 99600 PBMC. There was no significant decline over time in the proportion of PBMC containing provirus. Sequencing of a segment (287 bases) of the gag region of a West Australian FIV isolate (T90) revealed only slight nucleotide divergence from the North American Petaluma and PPR isolates and wider divergence from the Japanese TM2 clone.

Proviral DNA from four Australian isolates of feline immunodeficiency virus (FIV) was amplified by PCR and the nucleotide sequence determined for two conserved regions within gag (p 15/p 24) and pol (RT) genes. Comparison with the nucleotide and deduced amino acid sequence of two previously described U.S. isolates from California (Petaluma and PPR), and a third from Maryland (MD) as well as the Japanese isolate TM 2, revealed a close similarity between the Australian and Californian isolates with 95-97% nucleotide and 96-99% amino acid homologies. By contrast, the Maryland and Japanese isolates were more distantly related with only 84-87% nucleotide and 90-94% amino acid homology with either the Australian or Californian isolates. The relationship of the Australian FIV isolates to other domestic isolates as well as eight lentiviral isolates from wild felidae (panthers) published previously, was investigated further by constructing a phylogenetic tree based on the pol sequence. This revealed two subgroups of FIV, an Australian/Californian group and a less tightly clustered Maryland/Japanese group. These results suggest that the genomic variability of FIV is reflected by more than simply geographic distance. Furthermore, the relative genetic homogeneity found between Australian isolates suggest a shorter period of evolution of the virus in Australia than in North America.

Understanding the genesis of the block haplotype structure of the genome is a major challenge. With the completion of the sequencing of the Human Genome and the initiation of the HapMap project the concept that the chromosomes of the mammalian genome are a mosaic, or patchwork, of conserved extended block haplotype sequences is now accepted by the mainstream genomics research community. Ancestral Haplotypes (AHs) can be viewed as a recombined string of smaller Polymorphic Frozen Blocks (PFBs). How have such variant extended DNA sequence tracts emerged in evolution? Here the relevant literature on the problem is reviewed from various fields of molecular and cell biology particularly molecular immunology and comparative and functional genomics. Based on our synthesis we then advance a testable molecular and cellular model. A critical part of the analysis concerns the origin of the strand biased mutation signatures in the transcribed regions of the human and higher primate genome, A-to-G versus T-to-C (ratio ∼ 1.5 fold) and C-to-T versus G-to-A (≥ 1.5 fold). A comparison and evaluation of the current state of the fields of immunoglobulin Somatic Hypermutation (SHM) and Transcription-Coupled DNA Repair focused on how mutations in newly synthesized RNA might be copied back to DNA thus accounting for some of the genome-wide strand biases (e.g., the A-to-G vs T-to-C component of the strand biased spectrum). We hypothesize that the genesis of PFBs and extended AHs occurs during mutagenic episodes in evolution (e.g., retroviral infections) and that many of the critical DNA sequence diversifying events occur first at the RNA level, e.g., recombination between RNA strings resulting in tandem and dispersed RNA duplications (retroduplications), RNA mutations via adenosine-to-inosine pre-mRNA editing events as well as error prone RNA synthesis. These are then copied back into DNA by a cellular reverse transcription process (also likely to be error-prone) that we have called "reverse transcription-mediated long DNA conversion." Finally we suggest that all these activities and others can be envisaged as being brought physically under the umbrella of special sites in the nucleus involved in transcription known as "transcription factories."

The reactivity to a peptide from the HTLV-I polyprotein (FKLPGLNSR) and a similar sequence from myelin basic protein (MBP) (FKLGGRDSR) was examined in relation to the proposal that mimicry of MBP by HTLV-I could be involved in autoimmune responses in HTLV-I-associated myelopathy (HAM). It was found that rabbit antibodies raised against the HTLV-I peptide recognised both peptides, with a titre of 1/10240 to the HTLV-I peptide and 1/5220 to the MBP peptide. Human sera from HAM patients and a HTLV-I carrier without HAM showed slightly higher responses to the HTLV-I peptide compared to the responses from uninfected human sera. HAM patients had greater responses to the HTLV-I peptide than to the similar MBP peptide and an unrelated bovine MBP peptide. There was no recognition of the peptides by peripheral blood lymphocytes from HAM patients or a HTLV-I carrier without HAM. It was concluded that although cross-reactivity was demonstrated in rabbits and the HTLV-I peptide was recognised by sera from HAM patients, the epitope does not appear to evoke a mimicking response to the similar region in MBP. Hence it is not likely to be involved in the pathogenesis of HAM through molecular mimicry.

A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases.

The major histocompatibility complex (MHC) region contains several hundred genes in addition to the well-known HLA Class I and II loci. Some of these will explain MHC-disease associations. We have used paralogous and syntenic mapping to short list candidates and conclude that non-HLA genes present on human 6,1 and 19 and murine 17, 4 and 7 regulate immune responses and autoimmune disease. These genes are likely to be components of the early pre-HLA MHC and include tenascin and possibly notch inter alia.

Gamma inulin and Algammulin, two new adjuvants, were examined and compared with alum and Freund's Complete/Incomplete Adjuvant (FCA/FIA), for potentiation of cell-mediated immunity (CMI) and humoral immunity in sheep to a recombinant Taenia ovis antigen. The ability to protect sheep when challenged with live T. ovis eggs was also assessed. The results showed that gamma inulin and Algammulin induced a CMI response which was comparable to the FCA/FIA and alum groups and significantly higher than the control saline group. While gamma inulin, Algammulin and alum performed similarly and induced a significantly higher humoral immune response than the saline group. FCA/FIA elicited a much higher humoral immune response. Algammulin did not show the synergistic effect seen in mice and performed similarly to gamma inulin and alum alone. All the adjuvant groups induced significantly higher IgG1 and IgG2 levels than the saline group and they all favoured IgG1 production. When the sheep were challenged with live T. ovis eggs, at 25 weeks after primary immunization, the only group to show significant protection was the one which received FCA/FIA.

In an investigation of the evolution of feline immunodeficiency virus (FIV) in vivo, sequential isolates from a persistently infected cat were examined by direct sequencing following amplification of selected subgenomic regions by polymerase chain reaction (PCR). Three isolates, T90, T91, and T92, obtained over a three-year period revealed no changes to regions known to be conserved within gag and pol genes. Additionally, no change occurred within gag and pol in an isolate recovered from a second cat which was experimentally infected with T90. Changes were detected within an N-terminal region of the envelope glycoprotein gp 120 (env). These consisted of point mutations, some of which would result in amino acid substitutions and the predicted amino acid changes tended to cluster within variable domains. Inoculation of T90 into a second cat resulted in a different pattern of mutations than that observed for the three isolates from the first cat. In all cases, virus isolates derived from the same cat were much more highly related to each other (extent of env variation was 0.5-1.5%) than to isolates from other cats (10-12% env variation). The rate of change of FIV was estimated to be 3.4 x 10(-3) nucleotide substitutions per site per year for the env gene and less than 10(-4) nucleotide substitutions per site per year for the gag and pol genes, values concordant with that found for human immunodeficiency virus 1. Both nucleotide and amino acid changes in the gp 120 region were found to be directional, suggesting that selective pressures influence FIV envelope gene sequences.

From an examination of electroimmunoblots and peptide maps, a mouse monoclonal antibody to human myelin basic protein MBP was shown to react with the amino acid sequence Ala-Ser-Asp-Tyr-Lys-Ser which is located in the C-terminal half of MBP. Although a completely different immunization schedule was used by Sires et al. (1981) they obtained a monoclonal antibody reacting with the same determinant. In contrast to results with other monoclonal antibodies to globular proteins (Todd et al. 1982) this monoclonal antibody seems to react with a sequential rather than a topographical determinant.

An electroimmunoblot technique was used to detect various forms of myelin basic protein (MBP) in brain homogenates of 14 vertebrate species. Three antibodies were used to probe the immunoblots: a monoclonal anti-human MPB reacting with an antigenic determinant located at amino acid residues 131 to 136; a polyclonal anti-human MBP and a polyclonal anti-chicken MBP. Because no processing of the tissue is required prior to electrophoresis, in vitro artifacts are minimized. The 18.5 K form of MBP was present in all species except the shark. A 21.5 K MBP was observed in ovine, bovine, pig, rabbit, mouse, rat, monkey, but not in human, guinea pig, shark, toad and marsupial brains. A variant with a molecular weight between 17 K and 18 K was found in mouse, rat, bovine, human, monkey, pig, and chicken brains, and was the sole component in the shark brain. Marsupial brains had five or six forms of MBP between 14.5 K and 18.5 K.

Lactate accumalation was measured soon after decapitation in three adjacent lower spinal cord regions of rats with EAE. Results indicate that during EAE, and in correlation with the onset of clinical signal of both initial attack and shorth-term relapse, a differential focal increase in lactate accumulation occurs in rat spinal cord compared to Freund's Complete Adjuvant controls, with greater increase occurring in more caudal segments. A [14C]antipyrine method of estimating relative spinal cord blood flow failed to find evidence that the lactate accumulations were due to focal ischemia. Subsequent measurement of isotopic water and total protein increases in the same cord regions indicated that a slight but significant increase in vasogenic edema occurs in correlation with the increase in lactate accumulation and the onset of EAE clinical signs. The data are interpreted as lending support to a speculative theory of paralysis induced by edema during EAE, in which nerve root endoneurium is postulated as the functionally vulnerable site. More specifically, it is hypothesized that the ascending progression of clinical signs of EAE in rodents can be explained on an anatomica basis by progressive disturbance of the nodes of Ranvier in nerve root myelinated fibers.

Assays for total serum antibody, histamine sensitivity and the presence of reaginic antibodies were carried out on sheep repeatedly infected with first stage larvae of Lucilia cuprina. Effects of the sheep response on the larvae were monitored at the final infection and compared with control animals by the recovery of larvae and measurement of the wound caused by the larvae. Overall larval survival was not significantly different in the pre-infected group though wound sizes were smaller. Histamine sensitivity appeared to correlate with wound size only in the control group. Thus recent infection experience may lead to immune responses which override non-specific inflammatory events and cause smaller wound sizes. A sub-group of the pre-infected sheep had lower larval survival and smaller wound sizes than the other animals and this correlated with increased levels of reaginic antibody and lower total antibody levels. The results suggest a genetic basis for resistance to fly strike and the possible involvement of reaginic antibody in protective responses.

A solid phase competitive assay for human myelin basic protein (MBP) has been developed using a monoclonal antibody to MBP. The assay has been applied to the detection of antigenic peptides derived from MBP, the measurement of anti-idiotypic antibody and the detection of MBP in human serum.

Methylated amino acids are excreted in urine upon degradation of some tissue proteins. The urinary excretion ratios of NG,N′G-dimethylarginine (sym-DMA) and NG,NG-dimethylarginine (unsym-DMA) were studied in healthy adults and in patients with various diseases. The normal ratio of sym- to unsym-DMA in urine was 0.98 and ranged from 0.71 to 1.33; ratios were not significantly different in multiple sclerosis, cerebrovascular accident, cancer, and systemic lupus erythematosus. However, patients with liver disease, including chronic active hepatitis, were found on average to have a significantly altered ratio of 0.79, range 0.49–1.30, owing to an increase in the excretion of unsym-DMA. Hence measurements of the urinary excretion of dimethylarginine could become a useful aid in assessing recovery of liver cells in patients with chronic liver disease.

A procedure for producing monoclonal antibody to myelin basic protein (MBP) using in vitro immunization with MBP transferred to nitrocellulose is described. Following the separation of brain proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS—PAGE) and electrophoretic transfer of the electrophoretogram onto nitocellulose, the MBP band located by immunodetection was excised from the nitrocellulose, ground, and used as immunogen for in vitro stimulation of unprimed mouse spleen cells. While in vitro immunization with soluble MBP was able to generate many hybrids, all the wells in the fusions carried out with the immobilized MBP contained hybrids, 33 to 42% of which were positive to MBP. Among these, six were further characterized; all were IgM and all bound to epitopes common to the 18.5K and 21.5K MBP forms of several species. In view of its simplicity, this technique should have a wide application for the rapid production of monoclonal antibodies to selected proteins or their fragments present in small quantity or difficult to purify on a large scale.

A method for the electroimmunoblotting and immunodetection of peptides of less than 50 amino acid residues is described. Excellent resolution of a mixture of myelin basic protein (MBP) peptides was achieved by electrophoresis in a polyacrylamide stacking, urea-dodecyl sulphate minislab gel. Following electrophoresis, the peptides were transferred to various matrices and probed with monoclonal and polyclonal antibodies. Variables such as transfer time, membrane type, fixation and the amount of peptide loaded on the gel have been optimized and as a consequence native and synthetic peptides can now be visualized in gels and immunodetected on immobilizing matrices. This procedure is particularly suited to the analysis and identification of small MBP fragments arising in various neuropathological conditions as well as for the rapid characterization of antigenic determinants recognized by monoclonal and polyclonal anti-MBP antibodies.

Myelin basic proteins from human, bovine, porcine, rabbit and chicken central nervous systems were examined in aqueous solutions by proton n.m.r. at 400 MHz. Species comparisons and other techniques led to assignments of resonances to specific amino-acid residues. Titration effects associated with aspartic-acid sidechains were effective in identifying adjacent residues. Nuclear Overhauser difference spectra showed that the reverse turn previously suggested for the conformation of an isolated encephalitogenic nonapeptide (guinea-pig determinant) occurred also in the protein, thus supporting the concept of a special low-energy conformation associated with biological activity. The central region of the protein contains methyl groups in aliphatic sidechains with resonances having chemical shifts characteristic of strong shielding effects, thus supporting the idea that less extended secondary and tertiary structures occur in this region.

1. 1. Pig growth hormone (PGH) has been isolated by a method modified from that used for human growth hormone by Peckham (1967). 2. 2. The procedure involved ammonium sulphate precipitation, gel filtration on Sephadex G-Ioo and chromatography on DEAE-Sephadex A-50. Unlike previously described isolation methods, this procedure does not expose the hormone to extremes ofpH or to organic solvents. 3. 3. The isolated hormone appeared to be free from protein contaminants, but to be substantially contaminated with non-protein matter.

A previous hypothesis in which myasthenia gravis was explained by an immune response to acetylcholine receptors has been validated, and is here extended to cell receptors in general. Receptors on target cells, being accessible to circulating trophic hormones or transmitters, must also be accessible to antibodies which compete with the natural mediator for access to the site. To detect anti-receptor antibodies, physiological assay systems would be more sensitive than conventional immunological assays. Autoimmune responses to receptor sites would require a genetic predisposition to failure of immunological tolerance such as occurs in various autoimmune diseases. This hypothesis is supported by recent findings in hyperthyroidism and a type of insulin-resistant diabetes mellitus, and is applicable to other endocrinopathies, diseases in which dysfunction at receptor sites can be postulated, and regulatory functions within the immune system itself.

EAE in rodents has been the most widely-studied research analog of human demyelinating disease, especially multiple sclerosis. However, accumulating evidence indicates that classical, axon-sparing demyelination cannot be solely responsible for typical behavioral deficits during EAE. The puzzle of clinical pathologic correlation during EAE in rodents is reviewed, emphasizing the discrepancy between the consistent occurrence of “ascending” hind paralysis and the variability of histopathologic changes in the central nervous system (CNS). Several alternative hypotheses of the cause of paralysis during EAE are briefly discussed, including a recently-developed theory based on functional disturbance of peripheral nodes of Ranvier in nerve root myelinated fibers. It is argued that such a theory has definite advantages in explaining typical clinical signs of EAE in rodents.

A solid phase radioimmunoassay is described which employs 125I-protein-A to detect the presence of antibodies against a panel of cellular and soluble central nervous system (CNS) specific antigens coated onto polyvinylchloride Microtiter plates. Serum antibodies from rabbits immunized against myelin, myelin basic protein (MBP), glial acidic fibrillary protein (GFAP), astroglioma cells, and cerebellar cells were easily detected, and high specificity for each antiserum and antigen was also demonstrable. The assay is applicable to cerebrospinal fluid (CSF) from patients with neurological diseases to detect antibodies against CNS-specific antigens. The assay should be useful for examining cell lines derived from CNS tissue for the presence of brain proteins.

A rapid method for similarity searches (FASTP program) was used to identify similarities between a protein database and the human basic proteins from myelin [P2 protein and 17.2K, 18.5K, and 21.5K variants of myelin basic protein (MBP)]. From similarity scores, we concluded that none of the presently known proteins are in a family containing the MBPs. No new members were found for the lipid-binding family of which P2 is a member. Sequence similarities deemed relevant to the molecular mimicry hypothesis for virus-induced autoimmunity were identified in FASTP data with the aid of microcomputer programs. Several MBP/viral protein similarities were found that have not been reported previously. Of note because of their association with demyelinating conditions were proteins from visna and vaccinia. Similarity with visna was specific to the 21.5K and 20.2K MBPs. The most interesting new possibility for mimicry involving the P2 protein was between the influenza A NS2 protein and a sequence region of P2 thought to be neuritogenic in animals and mitogenic for lymphocytes from some patients with Guillain-Barre syndrome (GBS). This may have relevance for some cases of GBS associated with the 1976 U.S.A. swine flu vaccination program. Because FASTP reports only the best similarities between proteins, searches with FASTP may not have detected all the examples of mimicry present in the database. Searches might also be more effective if similarities could be scored on immunological rather than structural relatedness.

A solid phase radioimmunoassay (RIA) for the detection of antibodies to myelin basic protein (MBP) in sera has been developed employing MBP-coated flexible polyvinylchloride microtiter trays and [125I]protein-A as the radiolabel. [125I]Protein-A directly binds to the Fc region of serum IgG from several animal species and serves as an excellent reagent for detecting antibody. It can also be used to bind to a second antibody ligand, thereby making it useful even when it does not bind directly to the primary antibody Fc region. The use of one preparation of [125I]protein-A label allows sera from several species to be tested for antibodies to MBP simultaneously, thereby making this RIA technically simple, rapid and economical. This assay has been particularly useful in examining serum samples from animals with experimental autoimmune encephalomyelitis and hypomyelinogenisis congenita. In patients with multiple sclerosis low levels of antibody to MBP can be detected in cerebrospinal fluid (CSF) and acid-eluted extracts from brain plaque material; detection of low levels of antibody in human serum has not been possible due to non-specific binding of human serum Ig in this RIA.

Hind-limb motor function in adult female Lewis rats with experimental autoimmune encephalomyelitis (EAE) was investigated using an objective behavioral measurement of motor ability. Rats were pretrained to avoid falling from the external surface of a power-driven running wheel. The performance of EAE-group rats on the wheel was then compared with that of saline and adjuvant controls immediately prior to the onset of clinical signs of EAE, and within 3 days of apparent recovery from EAE. Results indicate no apparent hind-limb motor dificit in the absence of overt clinical signs of EAE, despite histological evidence of severe inflammatory lesions persisting in the central nervous system (CNS) at the time of the post-recovery test. The remarkably transient nature of motor impairment is discussed within the context of a continuing search for the underlying cause(s) of clinical signs of EAE.

The neurological signs induced by injection of tunicamycin are, in young adult rats, virtually identical to those typical of acute experimental autoimmune encephalomyelitis (EAE). Vasogenic exudation, of which the occurrence in the spinal cord of EAE rats has been shown to coincide with the onset of clinical signs, was investigated by quantitative electroimmunoblotting of central nervous system (CNS) tissue at various times following tunicamycin injection of young adult rats. Highly elevated levels of extravasated plasma proteins were observed in the spinal cord from 48 h after injection and, as in EAE rats, these increases coincided with the onset of neurological impairment. At 72 h post-injection, significant increases were also found in the brain of affected animals, albeit at much reduced levels. This is in contrast to previously reported findings in nursling rats where oedema was shown to be predominantly located in the brain. Quantitative electroimmunoblotting for myelin basic protein (MBP) in the CNS of tunicamycin-treated young adult rats indicated that, as in acute EAE, no extensive demyelination had occurred. These data provided further evidence that in both neurological diseases, vasogenic oedema of the spinal cord may be causally related to the appearance of neurological signs and suggested that its differential localization in the CNS may lead to differential neurological impairment.

Sheep were repeatedly exposed to a second stage excretory-secretory antigen preparation of Lucilia cuprina by intradermal injection (S group) or intranasal aerosol (I group) in an attempt to induce immunity to the larvae. Hypersensitivity responses to the injections were monitored and correlated with larval numbers at subsequent challenge. Intradermal injections showed that the immediate or IgE-mediated and the intermediate or Arthus response were the major skin hypersensitivity reactions to the larval antigens. At challenge there was no significant reduction in larval numbers between the S and the control group, however the Arthus reaction did show some correlation with larval recoveries in the S group. There was a significant reduction in larval numbers ( P

Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina . International Journal for Parasitology 16 : 69–75. Resistance to blowfly larvae infections developed in sheep exposed to at least four consecutive infections. Half of the sheep treated showed significant levels of resistance, the others remained susceptible. This resistance took the form of a decreased yield of third instar larvae in comparison to controls and sheep which remained susceptible. In addition an increased sensitivity to larvae developed, as shown by the area of wound obtained per maggot recovered, by the early appearance in resistant sheep of exudate from the infection site and by skin reactions to larval products. Radioimmunoassays demonstrated high levels of serum antibody against larval excretory/secretory antigens, though the response did not peak until after four infections. Resistant animals showed somewhat lower antibody titres than susceptible sheep. Consecutive infections of only 50 larvae failed to induce resistance to larger challenge infections. It is suggested that consecutive infections of larger numbers of maggots induce a hypersensitivity response which may effect larval survival especially of first and second instar maggots.

Scanning electron microscopy was used to determine whether first instar larvae were capable of physically damaging sheep skin and thus initiating blowfly strike without predisposing conditions. In addition, damage at the lesion site was monitored throughout the infection period. It was found that first instar larvae do not possess the mouth hooks seen in second and third instars. They do however have sets of spines within the oral cavity which, with the spines on the creeping welts, may be able to abrade the outermost skin cell layers. Damage to the epidermis was observed within the first 8 h after larval implantation on sheep. The dermis was exposed within the first 24 h and after this time the normal skin architecture was rapidly destroyed. At 72 h areas of wool were missing and the collagen matrix of the dermis was clearly visible. Predisposing conditions such as fleece rot or wounding were not necessary for strike initiation. It is suggested that a combination of physical irritation and abrasion together with enzymes released by larvae enable them to initiate formation of the strike lesion. Thus, the only mandatory predisposing condition for fly strike may be wetted wool and skin.

SUMMARY:Myelin basic protein (MBP) is an antigenic component of circulating immune complexes (CIC) in patients with multiple sclerosis (MS). Immune complexes were isolated from the sera by adsorption to Raji cells and then acid eluted. Final identification of MBP from Raji eluates was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by MBP radioimmunoassay (RIA) of gel eluates and by an immunoblot technique.

The amounts of anserine (β-alanyl-L-1-methylhistidine), carnosine (β-alanyl-L-histidine) and ophidine (β-alanyl-L-3-methylhistidine) were determined in various muscles and other tissues of pigs. The amount of the histidine dipeptides varied with age and muscle type. Ophidine accumulated in the longissimus dorsi at a rate of 25 nmol g−1 protein day−1 while the anserine concentration remained relatively constant. Thus, an estimate of the age of a pig can be obtained from the anserine: ophidine ratio in a sample of its skeletal muscle. Because the histidine dipeptides are unaffected by cooking, it is possible from an analysis of the dipeptides to monitor the type of lean pig meat used in preparing tinned hams. As the anserine:carnosine:ophidine ratio in pig meat is quite different from that in other species it is possible to distinguish between genuine and imitation hams.

The cell-mediated inflammatory component of experimental autoimmune encephalomyelitis (EAE) in mice is measured by the radioisotopic technique. Mice are challenged with autologous spinal cord homogenate in Freund's complete adjuvant and at various time intervals after such immunization given [ 125 I]5-iodo-2′-deoxy-uridine which is incorporated into the mononuclear cell pool. The degree of cell-mediated inflammation is determined by radiometry of the brain and spinal cord tissues. Increased radiolabelling is detected in the brains 2 days prior to the onset of clinical signs of EAE; increased radioactivity of the spinal cord is concomitant with clinical signs. This technique is useful in staging the extent of EAE and may prove to be a powerful tool in studying cell-mediated reactions in other autoimmune diseases.

An electroimmunoblotting technique was used with a monoclonal antibody to myelin basic protein (MBP) to assess demyelination in 3 defined regions of the spinal cord in rats with acute experimental autoimmune encephalomyelitis (EAE). A slight loss in MBP was detected only in the sacrococcygeal region of the spinal cord after the onset of clinical signs. In all 3 spinal cord regions studied, significantly elevated levels of albumin and IgG were detected during the course of EAE by the same technique. At the onset of clinical signs, the levels of IgG and albumin were highest in the more caudal regions of the spinal cord. As the clinical signs became more severe, IgG and albumin levels increased in the more cranial regions of the spinal cord. These changes thus correlated with the ascending progression of clinical signs typical of EAE in rats. These results provided added evidence that in rats affected with acute EAE, the clinical signs occur independently of demyelination and coincide with vasogenic edema.

The patterns of reactivity of antisera raised in rabbits to human myelin and to the basic protein of human myelin were studied by immunofluorescence, using frozen sections from autologous as well as heterologous central nervous system (CNS) and smears of myelin and a synaptosomal membrane fraction of CNS. Whole myelin induced antibodies which reacted with myelinated areas in frozen sections of the CNS in a homogeneous pattern, and with smears of myelin and the synaptosomal membrane fraction; this activity was absorbed from serum by myelin, by the water-insoluble fraction of myelin, but not by the basic protein of myelin. The basic protein of myelin induced antibodies which reacted with myelinated areas in the CNS in a finely speckled pattern, and with smears of myelin and the synaptosomal membrane fraction; this activity was absorbed from serum by myelin, by myelin basic protein and partly by histones. This evidence of cross-reactivity in immunofluorescence tests between the myelin basic protein and histones was corroborated by partial competitive inhibition between histone and the myelin basic protein in the radioimmunoassay for antibody to basic protein. An antigen similar to myelin basic protein is present in the synaptosomal membrane fraction of CNS and hence synaptosomes are potential reactive sites for the pathogenic immune response in experimental allergic encephalomyelitis.

A bovine encephalitogenic polypeptide fraction (BEP) from ox spinal cord was iodinated with 125I and 127I by the technique of Hunter and Greenwood.(5) Iodinated BEP retained its electrophoretic and chromatographic properties and encephalitogenic activity. Hydrolysis of 127I BEP showed that tyrosine and histidine were labelled. Delayed hypersensitivity reactions with BEP used as antigen performed on guinea pigs immunized with BEP trace labelled with 125I were positive. However the incidence of delayed hypersensitivity reactions on animals immunized with heavily iodinated 125I BEP was low, in spite of their normal encephalitogenic response. Foetal calf serum, the serum of adult animals and man and human cerebrospinal fluid appeared to contain a proteinase or peptidase capabel of digesting BEP. Proteolysis of supposedly sequestered autoantigens of myelin may be a normal means of preventing them from reaching the immunological system.

Isoelectric focusing was used to characterize proteolytic enzymes in homogenate and excretory-secretory preparations of the larvae of L. cuprina, the sheep blowfly. Zymogram overlays showed that the larvae produce a number of highly active proteases which have a wide range of isoelectric points and molecular weights. The alkaline and neutral pI proteases were inhibited by phenylmethyl-sulfonylfluoride, leupeptin and aprotinin; this indicated the presence of serine in the active site. Pepstatin and the metal chelating agent ethylenediaminetetraacetic acid had no effect Oil the activity of any of the proteases. Optimal pH for activity of the proteases was between 7 and 8. In addition, the proteases were found to be heat labile. Digestion of collagen fibrils confirmed the existence of collagenolytic activity in the excretory-secretory enzyme preparations. It is suggested that these enzymes may be involved in the nutrition of the larvae and in the pathogenesis of the lesion on the skin.

Myelin basic proteins from human, cow, pig, rabbit and chicken central nervous systems were studied in aqueous solution by proton n.m.r. at 400 MHz. Species comparisons and other techniques led to the assignment of resonances of 23 specific amino acid residues in the primary structure. Resonances from side chains of polar amino acids adjacent to aspartic residues could be assigned by anomalous effects of pH on the chemical shifts. The pK values of histidine side chains all fall in the range 6.0-6.9, and four specific histidine residues were assigned. The conformation of the protein in aqueous solution is that of an extended non-random polypeptide chain with regions of localized structure. Nuclear Overhauser difference spectra showed that a reverse turn similar to that previously suggested for an encephalitogenic nonapeptide isolated from the protein (guinea pig determinant) occurs also in the protein itself, thus supporting the concept of special low-energy conformations responsible for biological activity. Upfield chemical shifts of some side chain methyl groups from the central region of the primary structure suggest ring-current shifts due to higher-order structuring.